Powassan virus (POWV), a tick-borne flavivirus endemic to the USA, Canada, and Russia, has steadily increased in prevalence over the past decade. In a subset of cases, it leads to severe, often fatal, encephalitic disease. Furthermore, following recovery, long-term debilitating neurological sequelae occur in ~50% of individuals. No virus-specific therapeutic options currently exist, restricting treatment to supportive care. Monoclonal antibodies have been used clinically since 1986 for cancer, autoimmunity and infectious disease control and neutralizing monoclonal antibody therapy for POWV is one potential treatment approach. Nanobodies, or the variant domains (VHH) of heavy-chain only antibodies (HcAb) found in camelid species, have recently been adopted by the medical research community, with several currently in clinical trials for a variety of diseases and diagnostics. Nanobodies have low immunogenicity, high specificity, and exceptional stability and solubility, as well as demonstrated superior efficacy, safety, and utility, making them strong candidates for future therapeutics. Serological data suggests that many alpacas and llamas in the NE USA may be exposed to POWV during their lifespan, and therefore likely harbor neutralizing antibodies. This study combines virological, molecular biochemical, and epidemiological approaches to identify and characterize novel POWV nanobodies derived from naturally infected and immunized camelid species. In Aim 1, total sera from alpacas and llamas will be screened for POWV neutralization activity. Positive animals will be immunized with recombinant envelope protein in order to boost antibody responses. From animals whose sera show the highest neutralization potential, POWV- specific HcAb populations will be purified by affinity chromatography and the 15 kDa VHH domains isolated. Bone marrow will also be extracted from these same serologically positive animals and the VHH loci will be amplified by PCR. Utilizing a two-pronged identification strategy, VHH protein pools and VHH loci will be characterized by LC/MS and MiSeq high-throughput sequencing, respectively. Loci sequences will then be translated in silico and matched to the MS data using our program Llama-Magic, providing complete amino acid sequences for POWV-specific nanobodies. In Aim 2, recombinant nanobodies will be expressed in E. coli, purified, and tested in vitro for POWV binding and neutralization as well as cross reactivity to other flaviviruses. This will yield a panel of potent POWV-specific nanobodies that will be useful for research, diagnostic and therapeutic purposes. Future experiments could explore their therapeutic efficacy using murine and non-human primate models of POWV encephalitis.